1. Field of the Invention
The present invention relates to a novel mannanase produced from Celluosimicrobium sp. strain HY-13
2. Description of the Related Art
Cellulose and hemicellulose decomposing microorganisms are found in soil and compost related ecosystem, and digestive canals of herbivores and invertebrates. These microorganisms play an important role in biological transformation of biomass (Flint et al., 2008, Nat. Rev. Microbiol. 6, 121-131; Kim et al., 2009, Appl. Environ. Microbiol. 75, 7275-7279; Perez et al., 2002, Int. Microbiol. 5, 53-63).
Among structural polysaccharides found in plant biomass, β-1,4-mannan is the hemicellulose of softwood and is often found in various lignocellulose biomasses as heteropolysaccharide containing glucose and/or galactose units (van Zyl et al., 2010, Proc. Biochem. 45, 1203-1213). During the biodegradation of cellulose and xylan, mannan polysaccharide is also completely degraded by the cooperation of various hydrolases such as β-mannanase, β-mannosidase, β-glucosidase, α-galactosidase, and acetyl mannan esterase, according to the previous reports (Shallom and Shoham, 2003, Curr. Opin. Microbiol. 6, 219-228; Walker and Wilson, 1991. Bioresour. Technol. 36, 3-14).
β-1,4-mannanase is the endo-enzyme that degrades β-1,4-mannosidic binding in mannan polysaccharides randomly. Such glycoside hydrolase (GH) has the typical molecular structure composed of (β/α)8-barrel exhibiting double confront mechanism having anomer structure (Moreira and Filho, 2008, Appl. Microbiol. Biotechnol. 79, 165-178). β-1,4-mannanase in microorganism seems to be related to three GH family members, 5, 26, and 113, based on the amino acid sequence and structural/mechanical homology (cazy with the extension .org of the world wide web). Approximately 140 gene sequences of β-1,4-mannanase can be obtained from NCBI data-base. The homology among many genes encoding β-1,4-mannanase, which are originated from various bacteria and fungi, has been confirmed by genome analysis. Nevertheless, genetic and biochemical characteristics of β-1,4-mannanase originated from microorganism have not been disclosed as much as the characteristics of β-1,4-endoglucanases and β-1,4-xylanases.
Various aerobic and anaerobic cellulose decomposing microorganisms found in intestines of invertebrates and herbivores drew our attention recently as promising candidates capable of producing a novel GH enzyme having specific structural and functional characteristics (Brennan et al., 2004, Appl. Environ. Microbiol. 70, 3609-3617; Selinger et al., 1996, Anaerobe 2, 263-284). Even though it is presumed that an invertebrate such as earthworm that magnetizes soil organic matters has various cellulose decomposing microorganisms, most carbohydrolases have been confirmed to be the enzymes that degrade cellulose and xylan originated from rumen bacteria and insect intestine bacteria.
Two hemicellulose decomposing bacteria identified so far are Cellulosimicrobium sp. HY-13 (Kim et al., 2009, Proc. Biochem. 44, 1055-1059; Kim et al., 2009, Appl. Environ. Microbiol. 75, 7275-7279) and Streptomyces thermocarboxydus HY-15 (Kim et al., 2010, J. Mol. Catal. B: Enzym. 62, 32-39), which have been isolated from Eisenia fetida intestines. Extracellular endo-β-1,4-xylanase thereof was identified and molecularly confirmed. However, there have been no studies to disclose the characteristics of mannanase produced from the earthworm symbiotic microorganism at molecular level.
Thus, the present inventors identified the novel mannanase originated from Celluosimicrobium sp. strain HY-13, the earthworm symbiotic bacterium, and further disclosed the structural and biochemical characteristics of the mannanase along with the optimum conditions for the specific activity. The present inventors thereby completed this invention by confirming that GH5 β-1,4-mannanase identified to have high specific activity toward ivory nut mannan and locust bean gum could be effectively used as a feed efficiency enhancer.